U.S. patent application number 13/723881 was filed with the patent office on 2014-04-17 for method of treating clostridium difficile-associated diarrhea.
This patent application is currently assigned to OPTIMER PHARMACEUTICALS, INC.. The applicant listed for this patent is Optimer Pharmaceuticals, Inc.. Invention is credited to Farah Babakhani, Yu-Hung Chiu, Sherwood Gorbach, Thomas John Louie, Starr Louise Miller-Shangle, Alex Romero, Pamela Sears, Youe-Kong Shue, Robert Brian Walsh.
Application Number | 20140107054 13/723881 |
Document ID | / |
Family ID | 38004552 |
Filed Date | 2014-04-17 |
United States Patent
Application |
20140107054 |
Kind Code |
A1 |
Sears; Pamela ; et
al. |
April 17, 2014 |
Method of treating clostridium difficile-associated diarrhea
Abstract
A method of treating a disease or disorder caused by the
presence of a bacterium selected from the group consisting
Clostridium species, Staphylococcus species, Enterococcus species
and combinations thereof comprising administering to a patient in
need an effective amount of a mixture, which comprises tiacumicin
B, lipiarmycin A4, and at least one of other macrocyclic
compounds:
Inventors: |
Sears; Pamela; (San Diego,
CA) ; Shue; Youe-Kong; (TW) ; Miller-Shangle;
Starr Louise; (San Diego, CA) ; Walsh; Robert
Brian; (San Diego, CA) ; Babakhani; Farah;
(San Diego, CA) ; Chiu; Yu-Hung; (San Diego,
CA) ; Romero; Alex; (San Diego, CA) ; Gorbach;
Sherwood; (San Diego, CA) ; Louie; Thomas John;
(San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Optimer Pharmaceuticals, Inc.; |
|
|
US |
|
|
Assignee: |
OPTIMER PHARMACEUTICALS,
INC.
San Diego
CA
|
Family ID: |
38004552 |
Appl. No.: |
13/723881 |
Filed: |
December 21, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11585521 |
Oct 23, 2006 |
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13723881 |
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Current U.S.
Class: |
514/28 ;
536/7.1 |
Current CPC
Class: |
A61K 31/70 20130101;
A61K 31/7048 20130101 |
Class at
Publication: |
514/28 ;
536/7.1 |
International
Class: |
A61K 31/7048 20060101
A61K031/7048 |
Claims
1.-46. (canceled)
47. A pharmaceutical composition comprising a compound of Formula
IX (OP-1435) ##STR00008##
48. The pharmaceutical composition according to claim 47 further
comprising a compound of Formula II (tiacumicin B) ##STR00009##
49. The pharmaceutical composition according to claim 48 further
comprising a compound of Formula XIV (lipiarmycin A4)
##STR00010##
50. The pharmaceutical composition according to claim 48 further
comprising at least one compound selected from ##STR00011##
##STR00012##
51. A method of treating a disease or disorder caused by the
presence of Clostridium difficile comprising administering to a
patient in need an effective amount of the pharmaceutical
composition of claim 48.
52. A method of treating a disease or disorder caused by the
presence of Clostridium difficile comprising administering to a
patient in need an effective amount of the pharmaceutical
composition of claim 49.
53. A method of treating a disease or disorder caused by the
presence of Clostridium difficile comprising administering to a
patient in need an effective amount of the pharmaceutical
composition of claim 50.
54. The method according to any of claims 51-53 wherein the disease
is diarrhea.
55. The method according to any of claims 51-53 wherein the disease
is colitis.
56. The method according to any of claims 51-53 wherein the disease
is infectious diarrhea.
57. The method according to any of claims 51-53 wherein the disease
is Clostridium difficile-associated diarrhea.
58. The method according to any of claims 51-53 wherein the disease
is associated with the use of antibiotics or cancer chemotherapies
or antiviral therapy.
59. The method of any of claims 51-53 wherein the composition is
administered in an amount of about 50 mg to about 1000 mg one to
three times daily within three to fifteen days.
60. The method of any of claims 51-53 wherein the composition is
administered in an amount of about 100 mg to about 400 mg once or
twice daily.
61. The method of any of claims 51-53 wherein the composition is
administered in an amount of about 200 mg once or twice daily.
Description
RELATED APPLICATIONS
[0001] This application claims benefit from U.S. Provisional Patent
Application Ser. No. 60/729,135 which was filed on Oct. 21, 2005
and U.S. Provisional Application Ser. No. 60/749,641 which was
filed Dec. 12, 2005. This application is continuation-in-part
application of U.S. patent application Ser. No. 10/520,863 filed
Jan. 11, 2005, which claims benefit of International Application
No. PCT/US2003/021977 filed Jul. 15, 2003, which claims benefit of
U.S. Provisional Application Ser. No. 60/399,956 filed Jul. 29,
2002. This application is also continuation-in-part application of
International Application No. PCT/US05/16750 filed May 13, 2005,
which claims benefit of U.S. Provisional Application Ser. No.
60/570,697 filed May 14, 2004. This application is yet
continuation-in-part application of International Application No.
PCT/US05/02887 filed Jan. 31, 2005.
[0002] The disclosures of the above-reference applications are
incorporated by reference in their entirety herein.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention relates to the treatment of a disease caused
by the presence of a bacterium selected from the group consisting
of Clostridium species, Staphylococcus species, and Enterococcus
species and combinations thereof, in particular a disease caused by
the presence of a bacterium selected from the group consisting of
Clostridium difficile ("C. difficile"), Clostridium perfringens
("C. perfringens"), Staphylococcus aureus ("S. aureus") and
combinations thereof, more particular a disease caused by the
presence of C. difficile. The disease may be colitis,
pseudomembranous colitis, or diarrhea.
[0005] 2. Description of the Related Art
[0006] Antibiotic-associated diarrhea (AAD) is caused by toxin
producing strains of C. difficile, S. aureus including
methicillin-resistant Staphylococcus aureus (MRSA), and Clostridium
perfringens (C. perfringens). AAD represents a major economic
burden to the healthcare system that is conservatively estimated at
$3-6 billion per year in excess hospital costs in the U.S.
alone.
[0007] AAD is a significant problem in hospitals and long-term care
facilities and in the community. C. difficile is the most common
cause of AAD in the hospital setting, accounting for approximately
20% of cases of AAD and the majority of cases of
antibiotic-associated colitis (AAC). The rising incidence of C.
difficile associated diarrhea (CDAD) has been attributed to the
frequent prescription of broad-spectrum antibiotics to hospitalized
patients [Wilcox et al., Lancet 1996, 348: 767-8].
[0008] The most serious form of the disease is pseudomembranous
colitis (PMC), which is manifested histologically by colitis with
mucosal plaques, and clinically by severe diarrhea, abdominal
cramps, and systemic toxicity. The overall mortality rate from CDAD
is low, but is much greater in patients who develop severe colitis
or systemic toxicity. A recent study has shown that even when death
is not directly attributable to C. difficile, the rate of mortality
in CDAD patients as compared to case-matched controls is much
greater.
[0009] Diarrhea and colitis are caused by the elaboration of one or
more C. difficile toxins. The organism proliferates in the colon in
patients who have been given broad-spectrum antibiotics or, less
commonly, cancer chemotherapy. CDAD is diagnosed in approximately
20% of hospitalized patients who develop diarrhea after treatment
with such agents.
[0010] Current therapy for AAD or CDAD includes discontinuation of
implicated antimicrobial or chemotherapy agents, nonspecific
supportive measures, and treatment with antibiotics directed
against C. difficile. The most common antimicrobial treatment
options include vancomycin, and Metronidazole. Treatment of CDAD
with antibiotics is associated with clinical relapse of the
disease. Frequency of relapse is reported to be 5-50%, with a
20-30% recurrence rate being the most commonly quoted figure.
Relapse occurs with nearly equal frequency regardless of the drug,
dose, or duration of primary treatment with any of the antibiotics
listed above. The major challenge in therapy is in the management
of patients with multiple relapses, where antibiotic control is
problematic.
[0011] The two most commonly utilized specific therapies are
vancomycin and metronidazole, though vancomycin is the only drug
approved by the FDA for this indication. However, Vancomycin is not
recommended for first-line treatment of CDAD mainly because it is
the only antibiotic active against some serious life-threatening
multi-drug resistant bacteria. Therefore, in an effort to minimize
the emergence of vancomycin-resistant Enterococcus (VRE) or
vancomycin-resistant Staphylococcus aureus (VRSA), the medical
community discourages the use of this drug except when absolutely
necessary.
[0012] Metronidazole is recommended as initial therapy out of
concern for the promotion and selection of vancomycin resistant gut
flora, especially enterococci. Despite reports that the frequency
of C. difficile resistance may be >6% in some countries,
metronidazole remains nearly as effective as vancomycin, is
considerably less expensive, and can be used either orally or
intravenously. Metronidazole is associated with significant adverse
effects including nausea, neuropathy, leukopenia, seizures, and a
toxic reaction to alcohol. Furthermore, it is not safe for use in
children or pregnant women.
[0013] Although both agents are effective in treating the
infection, increasing rates of treatment failures and recurrence of
diarrhea in approximately 20% of patients that initially respond
are deficiencies of standard therapies. Therapy with metronidazole
has been reported to be an important risk factor for VRE
colonization and infection. In addition, the current treatment
regime is rather cumbersome, requiring up to 500 mg qid for 10 to
14 days. Thus, there is a need for better treatment for cases of
CDAD as well as for cases of other AAD and AAC.
[0014] Therefore, there is a need to develop a bactericidal drug
with a low propensity to generate resistance, having reduced or no
cross-resistance to existing antimicrobials and/or a prolonged
post-antibiotic effect.
SUMMARY OF THE INVENTION
[0015] The present invention provides a method of treating a
disease or disorder caused by the presence of a bacterium selected
from the group consisting of Clostridium species, Staphylococcus
species, Enterococcus species and combinations thereof comprising
administering to a patient in need an effective amount of a
mixture. The mixture comprises an effective amount of tiacumicin B
and an additional macrocycle selected from the group consisting
of:
##STR00001## ##STR00002## ##STR00003##
and combinations thereof. When the compound of formula XIV is
present, the mixture comprises about 0.1 to about 5% compound of
formula XIV.
[0016] Preferably, the mixture comprises at least 90% tiacumicin B
by weight. More preferably, the mixture comprises at least 95%
tiacumicin B by weight.
[0017] Preferably, the mixture comprises at least 1%, more
preferably, from about 2% to about 5%, of additional macrocycle by
weight.
[0018] Preferably, the mixture comprises about 0.1% to about 5%,
more preferably 0.3% to 3%, in particular 0.3% to 1.5%, especially
about 1%, lipiarmycin A4 by weight.
[0019] Preferably, when liapiarmycin A4 is present, the mixture
also comprises at least one of the following compounds:
##STR00004##
[0020] Preferably, the mixture exhibits an HPLC profile
substantially depicted at FIG. 5
[0021] Preferably, the disease or disorder treated in accordance
with the present invention is associated with C. difficile, C.
perfringens, S. aureus, and combinations thereof. More preferably,
the disease or disorder treated in accordance with the present
invention is associated with C. difficile.
[0022] Preferably, the disease treated in accordance with the
present invention is diarrhea or colitis, in particular diarrhea,
more particularly CDAD.
[0023] Preferably, the mixture in accordance with the present
invention is prepared by a process comprising: [0024] culturing a
microorganism in a nutrient medium to accumulate the mixture in the
nutrient medium; and [0025] isolating the mixture from the nutrient
medium; [0026] the nutrient medium comprises an adsorbent to adsorb
the mixture.
[0027] The nutrient medium preferably comprises 0.5-15% of the
adsorbent by weight. The absorbent is preferably an adsorbent
resin. More preferably, the adsorbent resin is selected from the
group consisting of Amberlite.RTM. XAD16, XAD16HP, XAD2, XAD7HP,
XAD1180, XAD1600, IRC50, and Duolite.RTM. XAD761. The microorganism
is preferably Dactylosporangium aurantiacum subspecies hamdenensis.
The nutrient medium comprises, based on weight, 0.2% to 10% of
glucose, 0.02% to 0.5% of K.sub.2HPO.sub.4, 0.02% to 0.5% of
MgSO.sub.4.7H.sub.20, 0.01% to 0.3% of KCl, 0.1% to 2% of
CaCO.sub.3, 0.05% to 2% of casamino acid, 0.05% to 2% of yeast
extract, and 0.5% to 15% of XAD-16 resin. The culturing step is
preferably conducted at a temperature from about 25 to about
35.degree. C. and at a pH from about 6.0 to about 8.0.
[0028] Preferably, the disease treated in accordance with the
present invention is associated with the use of antibiotics or
cancer chemotherapies or antiviral therapy.
[0029] In accordance with one preferred embodiment, the mixture is
administered in an amount of about 50 mg to 1000 mg, more
preferably 100 mg to 400 mg, in particular 200 mg, one to three
times daily, more preferably once or twice daily, in particular
twice daily, within three to fifteen days, in particular around ten
days. Oral administration is preferred.
[0030] The treatment of the present invention may allow for the
effective treatment of diarrhea diseases associated with
enterotoxigenic strains of C. difficile, S. aureus, and C.
perfringens without compromising systemic antibiotics and without
increasing vancomycin resistant enterococci (VRE) in the gut. The
present invention also reduces the presence of VRE in the gut.
[0031] Other objects and features of the present invention will
become apparent from the following detailed description considered
in conjunction with the accompanying drawings. It is to be
understood, however, that the drawings are designed solely for
purposes of illustration and not as a definition of the limits of
the invention, for which reference should be made to the appended
claims. It should be further understood that the drawings are not
necessarily drawn to scale and that, unless otherwise indicated,
they are merely intended to conceptually illustrate the structures
and procedures described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] In the drawings:
[0033] FIG. 1 shows the Phase 1B-MD Dosing schedule.
[0034] FIG. 2 shows the bacteroides count following treatment.
Pairs signed-ranks test, 2 tailed. For counts <3 log 10, a value
of 2.9 was used.
[0035] FIG. 3 shows the effect of Vancomycin therapy vs B. fragilis
group.
[0036] FIG. 4 shows the quantitative reduction of C. difficile
vegetative counts after treatment with MCC.
[0037] FIG. 5 is a typical HPLC profile of the mixture, which may
be used in the method of the present invention. 7
DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS
[0038] The definitions of certain abbreviations or terms used in
the present application are provided as follows:
[0039] AAD=antibiotic-associated diarrhea
[0040] ATCC=American Type Culture Collection
[0041] .sup.13C=carbon 13
[0042] CO.sub.2=carbon dioxide
[0043] N.sub.2=nitrogen
[0044] H.sub.2=hydrogen
[0045] TAPS=N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic
acid
[0046] MOPS=3-(N-Morpholino)propanesulfonic acid
[0047] CDAD=Clostridium difficile-associated diarrhea
[0048] CLSI=Clinical and Laboratory Standards Institute, formerly
NCCLS
[0049] ED.sub.50=effective dose to produce 50% response
[0050] HPLC=high performance liquid chromatography
[0051] IR=infrared spectroscopy
[0052] LLOQ=lower limit of quantification
[0053] MCC=Macrocycle-Containing Composition
[0054] MIC=minimum inhibitory concentration
[0055] MIC.sub.50=minimum inhibitory concentration to inhibit 50%
of bacterial strains tested
[0056] MIC.sub.90=minimum inhibitory concentration to inhibit 90%
of bacterial strains tested
[0057] MRSA=methicillin-resistant Staphylococcus aureus
[0058] NCCLS=National Commmittee for Clinical Laboratory Standards,
now CLSI
[0059] PMC=pseudomembranous colitis
[0060] VRE=vancomycin-resistant enterococci
[0061] VRSA=vancomycin-resistant Staphylococcus aureus
[0062] The term "antibiotic-associated condition" refers to a
condition resulting when antibiotic therapy disturbs the balance of
the microbial flora of the gut, allowing pathogenic organisms such
as enterotoxin producing strains of C. difficile, S. aureus and C.
perfringens to flourish. These organisms can cause diarrhea,
pseudomembranous colitis, and colitis and are manifested by
diarrhea, urgency, abdominal cramps, tenesmus, and fever among
other symptoms. Diarrhea, when severe, causes dehydration and the
medical complications associated with dehydration.
[0063] The term "MCC" refers to a preparation primarily containing
tiacumicin B with respect to the whole antibiotic substance (e.g.,
at least 90%, preferably 95%-98% by HPLC assay). MCC also comprise
a small amount (e.g., at least 1%, preferably 2%-5%) of tiacumicin
B related compounds, i.e., lipiarmycin A4 and at least one of
compound of formula III-XIV shown above. PCT application
PCT/US03/21977, having an international publication number of WO
2004/014295 A2, provides a process of making a mixture comprising
tiacumicin B. The entire content of this PCT application is
incorporated herein as reference. However, MCC intended exclusively
for use in non-humans may contain less than 80% of Tiacumicin B
(with respect to the whole antibiotic substance, by HPLC
assay).
[0064] The term "excipient" refers to an inert substance added to a
pharmacological composition to further facilitate administration of
a compound. Examples of excipients include but are not limited to,
calcium carbonate, calcium phosphate, various sugars and types of
starch, cellulose derivatives, gelatin, vegetable oils and
polyethylene glycols.
[0065] The term "halogen" includes F, Cl, Br and I.
[0066] The term "macrocycles" refers to organic molecules with
large ring structures usually containing over 10 atoms.
[0067] The term "18-membered macrocycles" refers to organic
molecules with ring structures containing 18 atoms.
[0068] The term "membered ring" can embrace any cyclic structure,
including carbocycles and heterocycles as described above. The term
"membered" is meant to denote the number of skeletal atoms that
constitute the ring. Thus, for example, pyridine, pyran and
thiopyran are 6 membered rings and pyrrole, furan, and thiophene
are 5 membered rings.
[0069] The term "MIC" or "minimum inhibitory concentration" refers
to the lowest concentration of an antibiotic that is needed to
inhibit growth of a bacterial isolate in vitro. A common method for
determining the MIC of an antibiotic is to prepare several tubes
containing serial dilutions of the antibiotic, that are then
inoculated with the bacterial isolate of interest. Following
incubation at appropriate atmosphere and temperature, the MIC of an
antibiotic can be determined from the tube with the lowest
concentration that shows no turbidity (no growth).
[0070] The term "MIC.sub.50" refers to the lowest concentration of
antibiotic required to inhibit the growth of 50% of the bacterial
strains tested within a given bacterial species.
[0071] The term "MIC.sub.90" refers to the lowest concentration of
antibiotic required to inhibit the growth of 90% of the bacterial
strains tested within a given bacterial species.
[0072] The term "patient" refers to a human or animal in need of
medical treatment. For the purposes of this invention, human
patients are typically institutionalized in a primary medical care
facility such as a hospital or nursing home. However, treatment of
a disease associated with the use of antibiotics or cancer
chemotherapies or antiviral therapies can occur on an outpatient
basis, upon discharge from a primary care facility, or can be
prescribed by a physician for home-care, not in association with a
primary medical care facility. Animals in need of medical treatment
are typically in the care of a veterinarian.
[0073] The term "pharmaceutically acceptable carrier" refers to a
carrier or diluent that is pharmaceutically acceptable.
[0074] The term "pharmaceutically acceptable salts" refers to those
derived from pharmaceutically acceptable inorganic and organic
bases. Salts derived from appropriate bases include alkali metal
(e.g., sodium or potassium), alkaline earth metal (e.g.,
magnesium), ammonium and N(C.sub.1-C.sub.4 alkyl).sub.4.sup.+
salts, and the like. Illustrative examples of some of these include
sodium hydroxide, potassium hydroxide, choline hydroxide, sodium
carbonate, and the like.
[0075] The term "pharmaceutical composition" refers to a mixture of
one or more of the Tiacumicins described herein, or physiologically
acceptable salts thereof, with other chemical components, such as
physiologically acceptable carriers and/or excipients. The purpose
of a pharmaceutical composition is to facilitate administration of
a compound to an organism.
[0076] The term "physiologically acceptable carrier" refers to a
carrier or diluent that does not cause significant irritation to an
organism and does not abrogate the biological activity and
properties of the administered compound.
[0077] The term "pseudomembranous colitis" or "enteritis" refers to
the formation of pseudomembranous material (i.e., material composed
of fibrin, mucous, necrotic epithelial cells and leukocytes) due to
inflammation of the mucous membrane of both the small and large
intestine.
[0078] The term "Tiacumicin" as used herein refers to a family of
compounds all of which comprise the 18-membered macrocycle shown
below in Formula I:
##STR00005##
[0079] The term "Tiacumicin B" as used herein refers to the
18-membered macrocycle shown below in Formula II:
##STR00006##
[0080] The term lipiarmycin A4 as used herein refers to the
18-membered macrocycle shown below in Formula XIV:
##STR00007##
[0081] In accordance with one embodiment of the present invention,
after multiple dose oral administrations, low MCC levels were
detected in plasma, most of which fell below the limit of
quantification. By contrast, fecal levels in both studies were
extremely high, exceeding 10,000 times the MIC.sub.90 (0.125
.mu.g/mL) versus C. difficile.
[0082] In accordance with one embodiment of the present invention,
recurrence of C. difficile-associated diarrhea can be inhibited in
a patient by administering MCC in an amount and for a duration
effective to inhibit recurrence of C. difficile but with a lack of
effect on normal gut flora in the patient.
[0083] In accordance with one embodiment, the daily oral dosage of
MCC for CDAD will range from between about 50 mg to about 1.0 grams
of active agent per day, preferably, from between about 100 mg to
about 600 milligrams per day. Generally, treatment will be
continued for a time period ranging from between about 3 to about
15 days. Greater or lesser amounts of drug and treatment intervals
may be utilized as required. For example, according to the results
of a clinical study hereinafter reported, a dosage of about 100-400
milligrams of MCC per day, over the course of from about ten days,
proved effective in treating CDAD with minimal clinical
recurrence.
[0084] In accordance with one embodiment of the present invention,
the mixture can be made by the following general process.
[0085] MCC-producing bacteria was grown in vessels ranging from
shake flasks to large "batch" fermenters. For producing substantial
quantities of MCC, submerged aerobic fermentation in tanks is
utilized. However, small amounts may be obtained by shake-flask
culture. For tank fermentation, it is preferable to use a
vegetative inoculum. The vegetative inoculum is prepared by
inoculating a small volume of culture medium with the spore form,
mycelial fragments, or a lyophilized pellet of the organism to
obtain a fresh, actively growing culture of the organism. The
vegetative inoculum is then transferred to a larger tank where,
after a suitable incubation time, the MCC antibiotic is produced in
much improved yield. It may be necessary to add small amounts of an
antifoam agent to large-scale fermentation media if foaming becomes
a problem.
[0086] The production proceeds in a control medium with other
additives/ingredients to improve the production. A
liquid-submerged, stirred-culture process is used for the
production of MCC. Fermentation is carried out at a temperature
range of 25.degree. C. to 37.degree. C. The consumption of the
carbon source is carefully monitored and an additional amount of
carbon source is added as needed. The pH of the fermentation is
preferably maintained between about 6.0 to about 8.0. MCC is
produced and accumulated between 3 to 15 days after inoculation of
the fermentation.
[0087] Commercially available adsorbent resins were found to
enhance the yield and recovery efficiency of MCC during the
fermentation. Adsorbents are preferably present in the range
between 0.5-15% by weight. MCC was recovered in exceptional yield
(>100 mg/L broth) from the fermentation broth by resin
absorption and eluted from the resin and mycelium by washing with
solvents of various polarities
[0088] MCC was first captured from the broth during fermentation
using adsorbent resins such as Amberlite resin (XAD-16). Upon
completion of fermentation, the solid mass (including the adsorbent
resin) is separated from the broth by sieving. The solid mass are
eluted with ethyl acetate then concentrated under reduced
pressure.
[0089] Upon completion of fermentation, the solid mass (including
the adsorbent resin) is separated from the broth by sieving. MCC is
eluted from the resin with organic solvents such as ethyl acetate,
methanol, acetonitrile or a mixture of two or more organic
solvents. The extract is then concentrated under reduced pressure.
This residue is further purified by trituration with low polarity
solvents such as hexanes, heptanes, methylcyclohexane, or by
partitioning between two phase solvent systems such as: ethyl
acetate/water; ethyl acetate/aqueous sodium chloride solution;
methanol/hexane, acetonitrile/hexane or other mixtures of two or
more solvents in various ratios and combinations or by column
chromatography eluting with an appropriate organic solvent system.
The current purification process of MCC is based on medium-pressure
reverse-phase (C-18) column using 50:50:1 CH.sub.3CN/H.sub.2O/AcOH
or 70:30:1, MeOH/H.sub.2O/AcOH as eluent. The fractions contain
desired MCC were washed with brine and the concentrated. The
residue was dissolved in ethyl acetate and washed with water and
organic layer was evaporated to dryness to provide a pale yellow
foam which was again washed with isopropyl alcohol and dried under
reduced pressure to yield a white powder. Combine fractions having
purity >88%. Concentrate fractions to one-half of original
volume. Filter precipitate and wash filter cake with water. The
solid was dried under high vacuum overnight to give a white powder
and analyzed by HPLC. Typically, the mixture comprising tiacumicin
B as major components ranged from 90% to 99%, lipiarmycin A4 (0.1%
to 5%), and at least one or more of the macrocycles of formula
III-XIV described above.
EXAMPLES
[0090] The following examples are provided by way of describing
specific embodiments of the present invention without intending to
limit the scope of the invention in any way.
[0091] The mixture used in the following examples is prepared in
accordance with the process of making described above. The
following table shows composition of several exemplary mixtures
made in accordance with the present invention.
API Mixture Profile
TABLE-US-00001 [0092] strain wild type wild type wild type wild
type wild type wild type wild type mutant mutant Trial fold 1A 1B
1B 1B 2A 2A 2A n/a (crude) 2B increased Lot # in MIC 92161001
92161901 92161902 92161903 93161001 93161901 93161002 F7502
B-0660041 relative to RRT % % % % % % % % % Compound tiacumicn B
0.32 -- 0.06 0.07 -- -- 0.06 -- -- 0.06 0.39 -- -- 0.07 -- -- -- --
-- 0.02 0.49 -- 0.19 0.13 -- -- -- 0.15 -- 0.05 0.71 -- -- -- -- --
-- -- 3.23 0.02 OP-1416 2-4x 0.75 0.48 0.28 0.16 0.17 0.32 0.08
0.12 0.49 0.35 0.79 0.11 0.09 0.07 0.06 0.05 0.08 -- 0.72 0.21 0.81
0.08 -- -- -- -- -- -- 1.01 0.08 OP-1415 8-16x 0.84 0.05 0.04 0.18
0.12 -- 0.87 -- 3.96 0.25 OP-1417 2-4x 0.86 -- -- -- -- -- -- -- --
-- 0.88 -- -- -- -- -- -- -- 3.20 -- 0.89 0.24 0.37 0.61 0.32 0.69
0.50 4.85 1.13 OP-1405 1-4x (lipiarmycin A4) 0.92 0.44 0.51 0.42
0.21 0.19 0.16 0.17 1.74 0.15 OP-1431 8x (tiacumicin F) 0.95 -- --
-- -- -- -- 0.08 0.63 0.06 OP-1432 8-16x (tiacumicin C) 0.96 0.27
0.44 0.65 0.35 -- -- 0.15 -- -- 0.98 0.25 -- -- -- -- -- -- -- --
1.00 95.54 97.26 96.40 98.78 98.16 98.55 98.36 73.61 95.6 OP-1441
1x (tiacumicin B) 1.03 -- -- -- -- -- -- 0.22 -- 0.22 1.04 0.29 --
-- -- -- -- -- -- -- 1.05 0.37 0.24 0.60 -- -- -- -- -- -- 1.07
0.43 -- 0.16 -- 0.08 -- -- -- 0.09 1.10 0.90 0.31 0.36 -- 0.26 0.10
0.10 0.37 -- OP-1434 >32x (tiacumicin A) 1.11 -- -- -- -- -- --
-- -- 0.81 1.13 -- -- -- -- -- -- -- -- 0.55 1.13 0.32 0.20 0.13 --
0.24 0.11 0.14 1.64 -- OP-1435 2x 1.14 0.19 -- -- -- -- -- -- -- --
1.19 0.04 -- -- -- -- -- -- 1.50 -- OP-1437 2x 1.23 -- -- -- -- --
-- -- 2.61 0.30 OP-1402 2-4x
[0093] The HPLC assay is conducted in accordance with the following
procedure.
TABLE-US-00002 Mobile Phase A: Add 2.0 mL of trifluoroacetic acid
to 2 L of HPLC water, filter and degas. Mobile Phase B: Add 1.0 mL
of trifluoroacetic acid to 2 L of Acetonitrile, filter and degas.
Column: 4.6 .times. 150 mm column that contains octyl silane
chemically bonded to porous silica or ceramic micro- particles 3 to
10 .mu.m in diameter (e.g., Zorbax Eclipse XDB-C8, 3.5 .mu.m).
Detector: 230 nm. Flow rate: About 1.0 mL/min. Injection volume:
About 10 .mu.L. Run time: About 10 .mu.L. Diluent: 100%
acetonitrile. Time (Min) % Mobile Phase A Mobile Phase B Gradient 0
60 40 program: 3.0 50 50 14.0 39 61 14.5 60 40
Note: Retention time of the mixture must be within 8-12
minutes.
[0094] Standard Preparation: Accurately weigh about 20 mg of the
mixture into a 100 mL volumetric flask, dissolve in and dilute to
volume with Diluent.
[0095] Sample Preparation: Accurately weight about 20 mg of the
mixture in a 100 mL volumetric flask. Add about 60 mL Diluent and
vortex to dissolve. Dilute to volume with Diluent and mix.
[0096] System Suitability: Chromatography the Standard preparation
and record the peak responses as directed under Procedure. The
relative standard deviation of tiacumicin B peak areas for five
replicate injections is NMT 2.0%, the tailing factor of tiacumicin
B areas is NMT 2.0.
[0097] Procedure: Inject about 10 .mu.L of Diluent. Separately
inject equal volumes (about 10 .mu.L) of Standard and Sample
preparations, record the chromatograms and measure the detector
responses for major peaks.
[0098] Relative Retention Time:
TABLE-US-00003 Related Substance RT ratio Compound of formula II
(tiacumicin B) 1.0 Compound of formula III 0.71 Compound of formula
IV 0.81 Compound of formula V 0.84 Compound of formula VI
(tiacumicin F) 0.92 Compound of formula VII (Tiacumicin C) 0.95
Compound of formula VIII (Tiacumicin A) 1.10 Compound of formula IX
1.13 Compound of formula X 1.19 Compound of formula XI 1.24
Compound of formula XII 1.39 Compound of formula XIII 1.48 Compound
of formula XIV (Lipiarmycin A4) 0.89
[0099] Calculations: Calculate the assay value using the following
formula:
Assay , % = R u R s .times. W std ( mg ) StdDil ( mL ) .times. P
.times. SmpDil ( mL ) W smp ( mg ) .times. WF .times. 100
##EQU00001##
[0100] Where: [0101] R.sub.u=tiacumicin B peak area obtained from
the assay preparation. [0102] R.sub.s=tiacumicin B peak area
obtained from the Standard preparation. [0103] P=Purity of
Reference standard, including water factor. [0104]
W.sub.std=Standard weight (mg) [0105] StdDil=Standard dilution
(mL). [0106] W.sub.smp=Sample weight (mg). [0107] WF=Sample water
factor.
[0108] Discard peaks originated from Diluent and calculate the
percentage w/w of individual and total related substances by the
formulae:
Individual related substance ( % w / w ) = R i R u .times. RF i
.times. 100 ##EQU00002##
[0109] Where: [0110] R.sub.i=Related substances peak area obtained
from the Sample Preparation. [0111] R.sub.u=tiacumicin B peak area
obtained from the Sample Preparation. [0112] RF.sub.i=Related
Substance response factor (RF.sub.i=1.0 for all related
substances.)
[0113] In addition, a typical HPLC profile of the mixture in
accordance with the present invention is shown in FIG. 5. The
compounds contained in the mixture, e.g., compounds of formula
II-XIV, may be found in the HPLC profile based on their RT ratio.
Par-101 in FIG. 5 represents tiacumicin B with RT ratio being
1.0.
[0114] The above mixture (50 mg) is then mixed with 100 mg Avicel
PH 102, FMC (microcrystalline cellulose) in a size 1 capsule
shell.
Example 1
Effect of Inoculum, pH, and Cations on the In Vitro Activity of MCC
Vs. Clostridium difficile
[0115] The MIC values measured for many antibiotics are known to be
affected by environmental variables such as pH, the concentration
of divalent cations such as calcium and magnesium, and the
bacterial density. The dependence of the antibacterial activity on
these factors is an important consideration, particularly for an
antibiotic that targets bacteria in the gut, where these parameters
can vary greatly with the diet and disease state. m
[0116] The sensitivity of the MIC to these environmental variables
may also be an important factor to consider when designing
methodology for future in vitro testing. The Clinical and
Laboratory Standards Institute, CLSI (formerly NCCLS) recommends
using Brucella agar supplemented with vitamin K.sub.1 and hemin for
Minimal Inhibitory Concentration (MIC) determination for anaerobes.
The level of divalent cations in this medium, however, is not
standardized. Moreover, the pH of the media used under anaerobic
glove box may also vary under different gas mixtures. Anaerobes are
typically incubated in a mixture of nitrogen, hydrogen, and carbon
dioxide, and the presence of CO.sub.2 will acidify the medium and
can be a significant source of variability. The inoculum size may
also be difficult to standardize given the variety of atmospheric
conditions available for anaerobic susceptibility testing
(H.sub.2/CO.sub.2 generator, evacuation/replacement method, or
anaerobic chamber). The anaerobic conditions available to each lab
will determine the duration of organism exposure to aerobic
atmosphere during bench top manipulations and anaerobic
equilibration, and thus affect culture viability and experimental
result.
[0117] In this study, we examined the effect on MIC of the level of
the divalent cations calcium and magnesium, pH (from 5-8), inoculum
density (over 3 orders of magnitude), and also the variability from
lot to lot of Brucella broth.
[0118] Materials and Methods
Bacterial Strains:
[0119] Laboratory strains of Clostridium difficile 9689, 700057,
43255, 17857 and Eubacterium lentum 43055 were obtained from
American Type Culture Collection (ATCC). All strains were streaked
onto brucella agar plates, supplemented with hemin, and vitamin K
from frozen stocks maintained at--at 78.degree. C. in 10% glycerol
prior to use.
MIC Testing:
[0120] Current CLSI procedures (4) for anaerobic broth and agar
dilution were used for MIC evaluation. Broth dilution is not a
validated method for MIC testing of Clostridium; however, due to
potential inaccuracy of measuring the pH of solid agar after
equilibration inside the anaerobic chamber, both methods were used
and compared for the assessment of pH effects.
Inoculum Density Effect on MIC Values:
[0121] The effects of inoculum density on susceptibility of C.
difficile to MCC and vancomycin were determined using the agar
dilution method (4). The inocula were prepared by first making a
suspension of .about.10.sup.8 cfu/mL and then serially diluting the
suspension by 10-fold factors to obtain a culture density range
between 10.sup.5-10.sup.8 cfu/mL, to give spot densities of
10.sup.2-10.sup.5 cfu/spot.
pH Effect on MIC Values:
[0122] The susceptibility of C. difficile to MCC was evaluated over
a pH range of 6-8 using both agar dilution and microbroth dilution
methods.
[0123] Using the agar dilution method, the MIC of MCC was
determined over a pH range of 6.2-8.0 against C. difficile strains
in two separate experiments. In order to achieve the desired
anaerobic pH for susceptibility testing, buffer (100 mM of
NaH.sub.2PO.sub.4 or TAPS
[N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic Acid]) was
added to media at pH 7 and 8, respectively. Even with strong
buffering, the pH shifted slightly following equilibration in the
anaerobic gas, and thus in some cases media was titered in ambient
air to above the desired anaerobic pH. The actual pH was always
confirmed following equilibration inside the anaerobic chamber.
Vancomycin, used as a control, was tested only at pH 7.
[0124] Using the broth microdilution method, the MIC values of MCC
and vancomycin were determined over a pH range of 6-8 against C.
difficile strains in 3 separate series. In the first series,
unbuffered Brucella broth was titrated in ambient air to obtain a
pH range from 5-9. However, anaerobic equilibration of media in the
glove box environment (10% H.sub.2/5% CO.sub.2/85% N.sub.2) lowered
the pH of the media, resulting in an anaerobic pH range from 5-7.5
(as tested using a portable pH meter with a flat-bottomed pH probe
calibrated with buffer standards outside the glove box, then
transferred inside). For subsequent experiments, buffer was added
to media to resist pH shifts caused by anaerobic equilibration. In
the second series, 10 mM buffer [NaH.sub.2PO.sub.4.H.sub.2O pH 7.0,
MOPS pH 8.0, or TAPS pH 9.0, pH values in ambient air) was added to
media with pH values greater than 6 to obtain a pH range from 6-7.6
after anaerobic equilibration. In the third series, the buffer
concentration was increased to 100 mM for pH treatments above 6 to
obtain an anaerobic pH range from 6-8.1.
Divalent Cation Concentration Effect on MIC Values:
[0125] The agar dilution method was used to determine the effect of
calcium and magnesium ion concentrations on susceptibility of C.
difficile strains to MCC. The level of divalent cations in the
Brucella broth as acquired from the manufacturer were determined by
the Laboratory Specialists, Inc. Additional amounts of divalent
cations were added (in the form of calcium or magnesium chloride)
in order to give calcium ion concentrations of 2.1, 3.0 and 5.7
mg/dL and magnesium ion concentrations of 3.3, 4.5, and 7.5
mg/dL.
Reproducibility of MCC MIC Values with Different Commercial Lots of
Media:
[0126] Using the CLSI agar dilution method, susceptibility of C.
difficile to MCC was also examined with three different commercial
lots of Brucella agar, from BBL (lot #30768960, 211086, and
3167036), supplemented with different lots of vitamin K (Sigma lot
#V-3501 and 0214010) and hemin (Sigma lot #072K1221 and
034K7656).
Results
Inoculum Density Effect on MIC Values:
[0127] Tables 1 and 2 demonstrate the effect of inoculum density on
the MIC of MCC and vancomycin against two strains of C. difficile
(ATCC 9689 and ATCC 700057). Susceptibility of both C. difficile
strains to MCC was unaffected by inoculum concentration from
10.sup.5-10.sup.8 cfu/ml (10.sup.2-10.sup.5 CFU/spot), as shown by
identical MIC values obtained for all inoculum concentrations
tested. The MIC of vancomycin, however, increased progressively
with increasing inoculum concentration, with the highest inoculum
density showing a fourfold increase in MIC over the lowest inoculum
density. These results demonstrate that inoculum density is not a
significant factor affecting the outcome of MCC susceptibility
testing of C. difficile.
TABLE-US-00004 TABLE 1 In vitro activity of MCC (.mu.g/mL) vs.
different inoculum densities of C. difficile ATCC 9689
(10.sup.2-10.sup.5 CFU/spot). Inoculum ATCC 9689 Density (cfu/ml)
CFU/spot MCC vanc 1.92 .times. 10.sup.8 1.92 .times. 10.sup.5 0.063
2 1.92 .times. 10.sup.7 1.92 .times. 10.sup.4 0.063 1 1.92 .times.
10.sup.6 1.92 .times. 10.sup.3 0.063 1 1.92 .times. 10.sup.5 1.92
.times. 10.sup.2 0.063 0.5
TABLE-US-00005 TABLE 2 In vitro activity of MCC (.mu.g/mL) vs.
different inoculum densities of C. difficile ATCC 700057
(10.sup.2-10.sup.5 CFU/spot). Inoculum Inoculum ATCC 700057 Density
(cfu/ml) Density (cfu/ml) MCC vanc 1.48 .times. 10.sup.8 1.48
.times. 10.sup.5 0.125 1, 2 1.48 .times. 10.sup.7 1.48 .times.
10.sup.4 0.125 1 1.48 .times. 10.sup.6 1.48 .times. 10.sup.3 0.125
1 1.48 .times. 10.sup.5 1.48 .times. 10.sup.2 0.125 0.5
pH Effect on MIC Values:
[0128] Table 3 depicts the effect of various pH values on
susceptibility of C. difficile to MCC as measured by agar dilution
method on two separate days. During the first run, the highest pH
treatment (pH 7.9) showed an 8-fold increase in MIC values over the
lower pH treatments (pH 6.2 & pH 7.2) for both strains of C.
difficile. When a confirmatory run was repeated at the highest pH
(pH 8.0), the MIC value remained high for both strains. No increase
in MCC MIC was observed between pH 6.2 and pH 7 for either
strain.
[0129] The increase in MIC values with pH did not consistently
correlate with increased growth, thus the effect of pH on MIC did
not appear to be merely due to the enhanced viability of the
organism at higher pH. The pH 7 treatment had less dense organism
spot growth relative to the pH 6.2 and pH 7.9 treatments.
TABLE-US-00006 TABLE 3 pH effects on agar dilution MIC values
(buffered medium) Anaerobic pH 7 7.9 8.0 6.2 100 mM
NaH.sub.2PO.sub.4 100 mM TAPS 100 mM TAPS Organism Drug Unbuffered
pH 7.2 (Air) pH 9.2 (Air) pH 9.2 (Air) ATCC 9689 MCC 0.063 0.063
0.5 1 Vancomycin 1 (pH 6.7) 4 ATCC 700057 MCC 0.125 0.125 1 2
Vancomycin 2 (pH 6.7) 4
[0130] Table 4, 5 and 6 represents MIC data from the broth
microdilution susceptibility method performed on three separate
days with pH ranges from 5 to 8.1. In the first series, in which
the medium was unbuffered, the MIC of MCC at pH 7.5 was 8.times.
greater than the MIC at pH 5.9 for both C. difficile strains (Table
4). The MIC at pH 5 could not be determined, because the organism
failed to grow at this pH. The buffered (10 mM) pH 7.6 treatment
showed 8-fold and 16-fold increases in MCC MIC over the pH 6
treatment for C. difficile ATCC 9689 & ATCC 700057,
respectively (Table 5). In the third, strongly buffered (100 mM)
series, similar results were seen with the highest pH treatment (pH
8.1) showing a 16-fold increase in MIC over the lowest pH treatment
(pH 6) for both organisms (Table 6). Vancomycin showed a similar
trend with the highest pH treatment producing MICs 4-8 fold greater
than the lowest pH treatment in all three experiments.
TABLE-US-00007 TABLE 4 pH effects on MIC using unbuffered media
Anaerobic pH (unbuffered) Organism Drug 5 5.9 6.6 7.1 7.5 ATCC 9689
MCC no growth .ltoreq.0.016 .ltoreq.0.016 0.063 0.125 MCC
.ltoreq.0.016 0.031 0.063 0.125 vanc 0.5, 1 0.5, 1 2 4 vanc 1 0.5,
1 2 4 ATCC 700057 MCC no growth 0.031 0.063 0.125 0.25 MCC 0.031
0.063 0.125 0.25 vanc 0.5 1 1 2, 4 vanc 0.5 1 1, 2 2, 4
TABLE-US-00008 TABLE 5 pH effects on MIC using weakly buffered
media (10 mM) Anaerobic pH 6.7 7.2 6 10 mM 10 mM 7.6 pH 6.0
NaH.sub.2PO.sub.4 MOPS 10 mM TAPS Organism Drug (Air) pH 7.0 (air)
pH 8.0 (Air) pH 9.0 (Air) ATCC MCC .ltoreq.0.016 0.031 0.063 0.125
9689 MCC .ltoreq.0.016 0.031 0.063 0.125 vanc 0.5 1 2 4 vanc 0.5 1
2 4 ATCC MCC 0.031 0.063 0.125 0.5 700057 MCC 0.031 0.063 0.25 0.5
vanc 0.5 1 2 4 vanc 0.5 1 2 4
TABLE-US-00009 TABLE 6 pH effects on MIC using strongly buffered
media (100 mM) Anaerobic pH 6.8 7.5 8 8.1 6 100 mM
NaH.sub.2PO.sub.4 100 mM MOPS 100 mM TAPS 100 mM TAPS Organism Drug
pH 6.0 (Air) pH 7.0 (Air) pH 8.0 (Air) pH 9.0 (Air) pH 9.5 (Air)
ATCC 9689 MCC .ltoreq.0.016, 0.031 0.031 0.125 0.25, 0.5 0.25, 0.5
MCC .ltoreq.0.016 0.031 0.125 0.25 0.25 vanc 1 1 4 >8 >8 vanc
0.5 1, 2 4 >8 >8 ATCC 700057 MCC 0.031, 0.063 0.063, 0.125
0.25 1 0.5 MCC 0.031 0.063, 0.125 0.25 0.5, 1 0.5 vanc 1 2 4 8 8
vanc 1 2 4 8 8
[0131] Assay plates at all pH treatments were also visually
examined for overall growth. In the first series, which utilized
unbuffered broth, overall culture turbidity increased with
increasing pH. The same trend was observed in the second series,
which utilized 10 mM buffered broth, except the culture turbidity
was the same for pH 7.2 and pH 7.6. In the third series, culture
turbidity was more equivalent across the pH treatments, with the
exception of pH 7.5, which was the most turbid.
[0132] Overall, with both methods of susceptibility testing and
across varying concentrations of buffer salts, the MIC values of
MCC and vancomycin increased with increasing pH for both strains of
C. difficile.
Divalent Cation Concentration Effect on MIC Values:
[0133] Measurement of the calcium and magnesium levels in
commercial Brucella broth showed calcium and magnesium ion
concentration of 21 and 33 mg/L, respectively. Various additional
amounts of divalent cations were added, and MCC MIC values for C.
difficile strains were tested at three different concentrations of
calcium ions (21, 30 and 57 mg/L) and three different
concentrations of magnesium ions (33, 45 and 75 mg/L). The MIC
values remained the same in all types of media. C. difficile 9689
had MIC value of 0.063 .mu.g/ml and C. difficile 700057 with MIC
value of 0.125 .mu.g/ml in media with varying concentrations of
cations. Vancomycin, which was tested as a control with
supplemented Brucella agar without any extra calcium or magnesium
as control during the experiments, demonstrated the expected MIC
value of 1 .mu.g/ml for all runs (Tables 7 and 8).
TABLE-US-00010 TABLE 7 In vitro activity of MCC in supplemented
Brucella agar with different divalent cation concentrations Calcium
concent in C. difficile C. difficile Brucella agar (ATCC 700057)
(ATCC 9689) Drug media (mg/L) MIC (.mu.g/mL) MIC (.mu.g/mL) MCC 33
0.125 0.063 45 0.125 0.063 75 0.125 0.063 Vancomycin 33 1 1
TABLE-US-00011 TABLE 8 In vitro activity of MCC in supplemented
Brucella agar with different divalent cation concentrations
Magnesium concent C. difficile C. difficile in Brucella agar (ATCC
700057) (ATCC 9689) Drug media (mg/L) MIC (.mu.g/mL) MIC (.mu.g/mL)
MCC 21 0.125 0.063 30 0.125 0.063 57 0.125 0.063 Vancomycin 21 1
1
MCC MIC Values with Different Commercial Lots of Media:
[0134] Three different lots of supplemented Brucella agar media
were used on three separate days to compare the activity of MCC
against C. difficile strains. The MIC assays were controlled by
testing the activity of the QC organism, Eubacterium lentum vs.
clindamycin which was within the CLSI (NCCLS) acceptable ranges,
i.e. 0.06-0.25 .mu.g/mL. Another control step for the MIC assays
was to include metronidazole and monitor its activity vs. C.
difficile strains, which in our laboratory has been shown to have
MIC values ranging between 0.25-0.5 .mu.g/mL. As shown in Table 9,
the activity of MCC vs. C. difficile was not affected by different
lots of supplemented Brucella agar. All controls demonstrated
activities within established ranges.
TABLE-US-00012 TABLE 9 In vitro activity of MCC tested with three
different lots of media MIC values (.mu.g/mL) Bacteria
Metronidazole Clindamycin MCC (ATCC #) Day 1 Day2 Day 3 Day 1 Day 2
Day 3 Day 1 Day 2 Day 3 C. difficile (9689) 0.5 0.5 0.5 4 4 4 0.25
0.25 0.125 C. difficile (43255) 0.5 0.5 0.5 8 4, 8 8 0.25 0.5 0.25
C. difficile (17857) 0.25 0.5 0.5 4 2 4 0.125, 0.25 0.25 0.125
Eubacterium 1 0.25 1 0.25 0.25 0.125 0.125, 0.25 0.25 0.06, 0.125
lentum (43055)
Conclusions
[0135] In contrast to vancomycin, the activity of MCC vs. C.
difficile was unaffected by inoculum concentrations, in the range
of 10.sup.2-10.sup.5 cfu/spot.
[0136] The susceptibility of C. difficile to MCC was unaffected by
cation concentrations (calcium ion in the range of 2.1-5.7 mg/dL
and magnesium concentration of 3.3-7.5 mg/dL), and by various
commercial lots of media.
[0137] The MIC values for both MCC and vancomycin increased with
increasing pH over a pH range of 6-8. The high MIC values at basic
pH may be due to deprotonated form of the phenolic hydroxyl groups
of both compounds above their pKa, where they form a charged
species that is expected to be less permeable to bacterial cells.
In contrast, below the pKa (7.22 for MCC), the antibiotics will be
mostly protonated, and thus should permeate the cell membrane more
efficiently.
[0138] Organism density generally increased with increasing pH; the
dependence of growth density, but not MIC, on pH was reduced in the
presence of buffering agents. Though organism density was
positively correlated with basicity in the absence of buffer, it is
unlikely that MIC trends are the result of the effect of pH on
organism density alone. This is because the same relationship
between MIC and pH was observed in buffered experiments where
organism density was more equivalent across pH treatments,
presumably due to the differential effect of buffer type on
organism growth.
Example 2
Safety, Pharmacokinetics and Outcomes of MCC in Healthy Subjects
and Patients with Clostridium difficile-Associated Diarrhea
(CDAD)
Phase 1B-MD.
[0139] Synopsis.
[0140] This was an oral, multiple-dose, double-blind, randomized,
placebo-controlled, dose escalation study conducted at the
University of Miami Division of Clinical Pharmacology, Miami, Fla.
Richard Preston, M.D. served as the Principal Investigator for this
trial. The tolerability and pharmacokinetics of multiple oral doses
of MCC were evaluated in a total of 24 healthy volunteer subjects.
The oral doses of MCC evaluated (in 3 groups of 8 subjects each,
with 6 active and 2 placebo) were 150, 300, and 450 mg (in
powder-filled capsules containing 50 mg of study drug) administered
daily after a morning breakfast for 10 consecutive days. Subjects
were dosed and monitored on a combined inpatient/outpatient basis.
Subjects were admitted to the research unit on Day 0 and again on
Day 9 of the 10-day dosing period, and stayed for up to 48 hours
after each admission. Subjects were discharged on Day 2 and Day 11
after completing the scheduled events and procedures. During the
outpatient period, subjects reported daily to the research unit for
dosing and stayed for 3 hours under observation.
[0141] Serial blood, urine, and fecal samples were collected at
various time points/intervals during the multiple dosing periods.
Plasma, urine, and fecal concentrations of MCC were determined for
pharmacokinetic analysis. A follow-up examination was scheduled on
Day 17 of each study period before subjects exited from the study.
Study subjects were closely monitored for the occurrence of any
adverse experiences or abnormal laboratory test findings throughout
the treatment periods and at the study follow-up. See, FIG. 1,
Phase 1B-MD Dosing schedule.
Phase 2A.
[0142] Synopsis.
[0143] This was a dose-finding study to select a safe and effective
dose of MCC. Subjects were randomized to receive either 100 (50 mg
every 12 hours), 200 (100 mg every 12 hours), or 400 (200 mg every
12 hours) mg/day for 10 days followed by clinical evaluation.
Subjects recorded all symptoms on daily diary cards. Particular
attention was to be given to stool frequency and consistency, the
presence of blood in the stool, and abdominal discomfort.
Laboratory assessments were performed at Screening for entry and at
End of Treatment (Day 10-12) or withdrawal (whichever was sooner).
Clinical observation and diary card evaluation were performed at
End of Treatment (Day 10-12). Patient interviews were conducted on
treatment Days 2 through 9, Day 17, and Day 52. For entry inclusion
criteria, assay for Clostridium difficile toxin was performed. For
subjects that failed to respond to MCC treatment, and in the event
of clinical recurrence, both C. difficile toxin assay and culture
were performed. Clinical, laboratory, and microbiological
assessments were also performed at exit for subjects that failed to
respond to treatment. Pharmacokinetic plasma samples were taken 0.5
hr prior to dosing and 2 hr after dosing on the first and last days
of dosing.
[0144] Key Inclusion Criteria.
[0145] Subjects were patients with C. difficile associated diarrhea
as defined by: 1) diarrhea (a change in bowel habits, with 3 or
more unformed bowel movements in 24 hours, or more than 6 loose or
watery stools within 36 hours.) and 2) presence of either toxin A
or B of C. difficile in the stool.
[0146] Key Exclusion Criteria
[0147] Subjects could not have 1) severe or life-threatening CDAD
2) life-threatening or serious disease unrelated to CDAD, 3)
concurrent use of: vancomycin, metronidazole, bacitracin, or
related drugs. (If the Investigator felt the clinical imperative to
begin treatment before knowing the laboratory result for stool
toxin, up to 24 hours, but no more than 3 doses, of treatment with
metronidazole and/or vancomycin was to be allowed.); any drugs used
for the treatment of CDAD; or other antibiotics 4) history of
ulcerative colitis or Crohn's disease and multiple recurrences
(defined as more than one recurrence) of CDAD within the past three
months. (Subjects with a single recurrence of CDAD were permitted
to enroll.)
Schedule of Events
TABLE-US-00013 [0148] TABLE 10 Schedule of Evaluation Procedures in
the phase 2A study Day 1 Treatment End of Screen/ Days 2 Treatment
Completion Day 52 Assessments Enrollment through 9 or Day 10 Day 17
Follow-up Informed Consent X Inclusion/Exclusion X Medical History
X Physical Examination X X Vital Signs X X 12-Lead ECG X X Clinical
Laboratory Tests X X Stool Sample X X PK Blood Sampling.sup.a X X
Fecal PK Sampling X Adverse Events X X X Concomitant Medication X X
X X X Pregnancy Test X Diary Card Review X X X Study Medication
Administration X X X Subject Interview X X X X X .sup.aBlood
samples for pharmacokinetics taken 0.5 hr prior to and 2 hr post
administration on the first and last days of dosing
[0149] Endpoints.
[0150] At the end of therapy, the investigator determined if the
subject had been cured of failed. In addition, the time to
resolution of diarrhea (defined as resolution to <3 loose or
watery stools per day) and the complete relief of symptoms of CDAD
by day 10 of therapy (complete relief was resolution to total
stools per day, whether loose or firm; and absence of fever,
elevated white blood cells, or abdominal pain) were tracked as
primary endpoints, and recurrence within 6 weeks following therapy
(recurrence of diarrhea, defined as 3 or more loose/watery stools
per day, with a positive toxin test) was tracked as a secondary
endpoint.
Analysis
Safety Population:
[0151] The safety population was to include all randomized subjects
who received at least one dose of study medication and had safety
information available.
Efficacy Population:
[0152] Clinical success or failure was determined in patients
treated per protocol.
[0153] The population analyzed for time to resolution of diarrhea
and complete relief of symptoms was the modified intent to treat
population (mITT), consisting of all randomized subjects who
received at least one dose of study medication, had a history of
diarrhea, and had 3 or more loose stools in 24 hours and a positive
C. difficile toxin at baseline.
[0154] Time to resolution of diarrhea was defined as time (in days)
from the first dose of study medication to the resolution of
diarrhea; time to resolution of diarrhea was compared among the
three treatment groups. The cessation day of diarrhea was defined
as the first day that <3 unformed stools (watery or loose)
within a 24 hour period occurred and was sustained for the duration
of treatment up to study Day 10. Resolution of diarrhea was
assessed during a 10 to 12 day period utilizing the subject diary
data.
Complete Relief of Symptoms of CDAD:
[0155] Complete relief of symptoms of CDAD was defined as
resolution to bowel movements per day (as recorded on the patient
diary) without other associated signs/symptoms such as fever
(.gtoreq.37.7.degree. C.), abdominal pain (no response on diary)
and elevated WBC (normal laboratory range of WBC) by Day 10 of the
study. If any variable was missing, this outcome was considered
unknown.
Clinical Recurrence Rate:
[0156] Clinical recurrence was defined as .gtoreq.3 unformed stools
(loose or watery) and a positive stool for C. difficile toxin A or
B within 6 weeks posttreatment.
Results
Enrollment and Demographics
[0157] The following sections summarize the enrollment and
demographic characteristics of the study populations in the phase
1B-MD and 2A trials. A total of 24 healthy subjects were enrolled
for the phase 1B-MD study. Alternate male and female subjects were
enrolled to provide an even split between the sexes. Subjects
ranged in age from 38-62 years (average 51.6.+-.7.5 yr), in weight
from 55.5-90 kg (average 71.5.+-.9.2 kg), and in height 147-183 cm
(average 164.8.+-.10.8 cm.)
[0158] In the phase 2B study, a total of 49 subjects were enrolled.
One subject withdrew consent and was dropped from the study prior
to receiving any study drug, and was not evaluable for either
safety or efficacy. One subject (400 mg dosing group) had >6
bowel movements in 36 hours, but <3 bowel movements in the prior
24 hours, and could not be evaluated for time to resolution of
diarrhea but was evaluable for clinical response and safety
analyses. Three patients were discontinued after 1 or 2 doses due
to removal of consent (1 subject, 100 mg dosing group), requirement
for additional antibiotics for pneumonia (1 subject, 100 mg dosing
group), or inability to take study medication (1 subject, 200 mg
dosing group). Subject demographics are listed in Table 11.
TABLE-US-00014 TABLE 11 Summary demographics for the Phase 2A
study; demographics for the 48 subjects in the population evaluable
for safety are shown. MCC MCC MCC 100 mg/Day 200 mg/Day 400 mg/Day
All Subjects (N = 16) (N = 16) (N = 16) (N = 48) Sex Female 10
(62.5%) 11 (68.8%) 9 (56.3%) 30 (62.5%) Male 6 (37.5%) 5 (31.3%) 7
(43.8%) 18 (37.5%) Race Caucasian 14 (87.5%) 15 (93.8%) 14 (87.5%)
43 (89.6%) Black 1 (6.3%) 1 (6.3%) 0 (0.0%) 2 (4.2%) Asian 0 (0.0%)
0 (0.0%) 1 (6.3%) 1 (2.1%) Hispanic 0 (0.0%) 0 (0.0%) 0 (0.0%) 0
(0.0%) Other.sup.a 1 (6.3%) 0 (0.0%) 1 (6.3%) 2 (4.2%) Age (Yrs)
Mean .+-. SD 56.3 .+-. 17.78 53.1 .+-. 22.97 55.3 .+-. 17.69 54.9
.+-. 19.26 Median 54.5 55.5 56.0 56.0 Range 28.0-89.0 18.0-88.0
18.0-90.0 18.0-90.0 Weight (Kg) Mean .+-. SD 69.2 .+-. 14.00 68.4
.+-. 11.46 67.5 .+-. 13.56 68.4 .+-. 12.82 Median 69.3 66.0 65.2
66.0 Range 38.0-89.0 52.0-96.0 40.0-88.2 38.0-96.0 Height (cm) Mean
.+-. SD 163.8 .+-. 15.52 166.4 .+-. 9.48 166.2 .+-. 13.18 165.5
.+-. 12.80 Median 162.1 170.0 163.8 165.0 Range 122.0-187.5
150.0-178.0 142.0-193.0 122.0-193.0 Calculated Body Mass
Index.sup.b Mean .+-. SD 25.8 .+-. 3.89 24.9 .+-. 4.50 24.3 .+-.
2.52 25.0 .+-. 3.68 Median 25.0 24.0 24.5 25.0 Range 17.0-34.0
17.0-32.0 20.0-28.0 17.0-34.0 NOTE: values represent number of
subjects unless otherwise indicated. .sup.aOther includes: East
Indian, Indian. .sup.bCalculated body mass index is defined as
(weight in kg)/(height in meters).sup.2.
Efficacy
[0159] In the clinical evaluation of treatment success or failure
at the end of therapy, two patients in the low dosing group (2/14),
2 patients in the mid dosing group (2/15), and no patients in the
top dosing group (0/16) were considered treatment failures by the
investigator. Among the subjects (n=41) that were treatment
successes, CDAD recurred in one subject (1/12) in the 100 mg/day
dosing group and one subject (1/16) in the top dosing group, for a
recurrence rate of 2/41 (5%) overall. Both recurrences occurred
approximately 1 month following the end of therapy.
TABLE-US-00015 TABLE 12 Rates of clinical cure and recurrence in
the population treated per protocol. MCC MCC MCC 100 mg/Day 200
mg/Day 400 mg/Day N % N % N % Total 14 100 15 100 16 100 Treatment
success 12 86 13 87 16 100 Treatment failure 2 14 2 13 0 0 Clinical
recurrence.sup.a 1 8.3 0 0 1 6.3 .sup.aRecurrence of toxin-posivite
diarrhea within 6 weeks post-treatment, evaluated in patients that
were clinical successes.
[0160] The time to resolution of diarrhea was defined as the time
for the patient to resolve to less than 3 unformed stools per day,
according to the patient's diary card. In the mITT population, the
median time to relief was 5.5 days, 3.5 days, and 3.0 days for the
MCC 100 mg/day, 200 mg/day and 400 mg/day treatment groups,
respectively. The mean time to resolution of diarrhea in days was
6.3.+-.3.66 days in 100 mg/day-treated subjects, 4.8.+-.3.56 days
in 200 mg/day-treated subjects, and 3.6.+-.2.03 in 400
mg/day-treated subjects. There was no statistically significant
difference in time to resolution of diarrhea between the 100 mg/day
and 200 mg/day treatment groups, and between the 200 mg/day and 400
mg/day treatment groups; however the difference between the 100
mg/day and 400 mg/day treatment groups approached statistical
significance (p=0.0506 Kaplan Meier estimate and p=0.0503
Kruskal-Wallis test).
TABLE-US-00016 TABLE 13 Time to Resolution of Diarrhea (mITT
population), defined as time to resolve to <3 unformed bowel
movements per day (according to the patient's diary card) MCC 100
mg/ MCC MCC Day 200 mg/Day 400 mg/Day P-Value N 16 16 15 N
(Resolved Diarrhea) 10 12 14 N (Censored: Did not 4 3 1
resolve).sup.a N (Censored: Dropped 2 1 0 from study) N (Censored:
Total) 6 4 1 Median Time (Days).sup.b 5.5 3.5 3.0 P-Value.sup.c
0.1912 MCC 100-MCC 200.sup.c 0.2901 MCC 100-MCC 400.sup.c 0.0506
MCC 200-MCC 400.sup.c 0.6143 .sup.aSubjects whose diarrhea was not
resolved to <3 loose stools/day by day 10 .sup.bKaplan-Meier
estimates .sup.cP-value obtained from generalized Wilcoxon
Test.
[0161] Complete relief of symptoms of CDAD by the end of treatment,
defined as .ltoreq.3 total bowel movements per day (whether formed
or unformed, as recorded on the patient's diary card), and no
fever, elevated WBC count, or abdominal pain (according to response
on patient diary card) by the 10.sup.th day of the study, is shown
in Table 14. Complete relief was achieved by 37.5% of the 100
mg/day treatment group, 50.0% of the 400 mg/day treatment group,
and 86.7% of the 400 mg/day treatment group. It is worth noting
that most patients that did not have complete relief by day 10 were
nevertheless treatment successes, had resolution of symptoms by day
17, and did not require further treatment. Three patients that
dropped from the study (one for removal of consent, one for the
requirement of exclusionary antibiotics, and one for the inability
to take oral medications) are also listed as having no complete
relief.
TABLE-US-00017 TABLE 14 Complete Relief of Symptoms of CDAD by end
of therapy in the mITT population, defined as resolution to
.ltoreq.3 total bowel movements per day (formed or unformed, as
noted on the patient's diary card) without other associated
signs/symptoms such as fever, abdominal pain, and elevated WBC by
Day 10 of the study MCC MCC MCC 100 mg/ 200 mg/ 400 mg/ Day Day Day
n % n % n % Complete Relief 6 (37.5) 8 (50.0) 13 (86.7) No Complete
Relief 9 (56.3) 6 (37.5) 2 (13.3) Required further treatment 2
(12.5) 2 (12.5) 0 (0) Required no further 5 (31.3) 3 (18.8) 2
(13.3) treatment Dropped from study 2 (12.5) 1 (6.3) 0 (0) Unknown
1 (6.3) 2 (12.5) 0 (0.0)
[0162] Only 2 subjects (1 subject in the 100 mg/day treatment group
and 1 subject in the 400 mg/day treatment group) experienced
clinical recurrence.
Safety
[0163] In the phase 1B-MD study, MCC was well tolerated by all
subjects at all doses. Fourteen adverse events were reported, 7 in
the 150 mg group, 2 in the 450 mg group, and 5 in the placebo
group. The adverse events are summarized as follows: headache (2),
dizziness (1), weakness (1), fatigue (1), nasal congestion (1),
difficulty swallowing (1), pharyngitis (1), conjunctivitis (1),
upper respiratory infection (2), rash (1), and pruritis (1). No
subjects receiving MCC had adverse events considered to be
drug-related.
[0164] In the phase 2A study, as shown in Table 15, 4/16 (25.0%)
subjects in the 100 mg/day treatment group, 4/16 (25.0%) subjects
in the 200 mg/day treatment group, and 1/16 (6.3%) subjects in the
400 mg/day treatment group, reported at least one AE during the
study. The highest frequency of AEs was reported in the infections
and infestations body system in the 100 mg/day treatment group
(3/16; 18.8% subjects). There were 2/16 (12.5%) subjects who
reported vascular disorders in the 100 mg/day treatment group and
2/16 (12.5%) subjects who reported gastrointestinal disorders in
the 200 mg/day treatment group.
TABLE-US-00018 TABLE 15 Incidence of adverse events in the safety
population of the 2A study, summarized by system organ class and
preferred term MCC MCC MCC 100 mg/ 200 mg/ 400 mg/ Day Day Day
System Organ Class (N = 16) (N = 16) (N = 16) Preferred Term n (%)
n (%) n (%) Total subjects with adverse 4 (25.0) 4 (25.0) 1 (6.3)
events Cardiac disorders 1 (6.3) 0 (0.0) 0 (0.0) Cardiac failure
congestive 1 (6.3) 0 (0.0) 0 (0.0) Gastrointestinal disorders 0
(0.0) 2 (12.5) 0 (0.0) Gastrointestinal haemorrhage 0 (0.0) 1 (6.3)
0 (0.0) Pancreatitis chronic 0 (0.0) 1 (6.3) 0 (0.0) General
disorders and 1 (6.3) 1 (6.3) 0 (0.0) administration site
conditions Chest pain 1 (6.3) 1 (6.3) 0 (0.0) Infections and
infestations 3 (18.8) 1 (6.3) 0 (0.0) Bronchitis 1 (6.3) 0 (0.0) 0
(0.0) Infection 1 (6.3) 0 (0.0) 0 (0.0) Pneumonia 1 (6.3) 0 (0.0) 0
(0.0) Staphylococcal sepsis 0 (0.0) 1 (6.3) 0 (0.0) Urinary tract
infection 1 (6.3) 0 (0.0) 0 (0.0) Injury, poisoning and procedural
0 (0.0) 1 (6.3) 0 (0.0) complications Fall 0 (0.0) 1 (6.3) 0 (0.0)
Metabolism and nutrition 0 (0.0) 0 (0.0) 1 (6.3) disorders Fluid
overload 0 (0.0) 0 (0.0) 1 (6.3) Musculoskeletal and connective 1
(6.3) 0 (0.0) 0 (0.0) tissue disorders Pain in extremity 1 (6.3) 0
(0.0) 0 (0.0) Nervous system disorders 0 (0.0) 1 (6.3) 0 (0.0)
Cerebral haemorrhage 0 (0.0) 1 (6.3) 0 (0.0) Renal and urinary
disorders 1 (6.3) 0 (0.0) 0 (0.0) Nephrolithiasis 1 (6.3) 0 (0.0) 0
(0.0) Respiratory, thoracic and 1 (6.3) 0 (0.0) 0 (0.0) mediastinal
disorders Dyspnoea 1 (6.3) 0 (0.0) 0 (0.0) Vascular disorders 2
(12.5) 0 (0.0) 0 (0.0) Hypotension 2 (12.5) 0 (0.0) 0 (0.0) NOTE:
Percentages are the proportions of subjects within that
category.
[0165] Five subjects were reported as having SAEs during the study
(Table 16). In the 100 mg/day treatment group, one subject had
diarrhea of moderate severity and another subject had severe
exacerbation of congestive heart failure (CHF). In the 200 mg/day
treatment group, one subject had severe staphylococcal sepsis and a
severe cerebral hemorrhage, another subject had a gastrointestinal
hemorrhage of moderate severity, and a third subject had chest pain
of moderate severity. No subject in the MCC 400 mg treatment group
had an SAE. All SAEs were considered to be unrelated to study
drug.
TABLE-US-00019 TABLE 16 Incidence of serious adverse events in the
safety population of the 2A study Total Duration Treatment of
Duration Relationship Subject Age Therapy Adverse Event Study of AE
to Study Number (Yrs) Sex Race (Days).sup.a (Preferred Term)
Day.sup.b (Days) Severity Drug.sup.c Outcome MCC100 mg/day 314 34
Male Caucasian 10 Diarrhea 33 3 Moderate Not Related Recovered
Without Sequelae 400 52 Male Black 10 Cardiac Failure 39 12 Severe
Not Related Recovered Congestive With Sequelae MCC 200 mg/day 200
85 Female Caucasian 10 Staphylococcal 10 7 Severe Not Related Not
Yet Sepsis Recovered.sup.d Cerebral 10 7 Severe Not Related Not Yet
Haemorrhage Recovered.sup.d 208 71 Female Caucasian 10
Gastrointestinal 15 14 Moderate Not Related Recovered Haemorrhage
Without Sequelae 304 59 Female Caucasian 11 Chest Pain 23 6
Moderate Not Related Recovered Without Sequelae .sup.aDate of last
dose of study medication minus date of first dose of study
medication plus one. .sup.bStudy day is calculated as follows: date
of onset minus date of first date of study medication plus one.
.sup.cBased on Investigator's assessment. .sup.dSubject died.
Pharmacokinetics
Plasma Concentration Data
[0166] In the phase 1B-MD study, after multiple dose oral
administrations, plasma concentrations of MCC were mostly below the
limit of quantification across the dose range
[0167] Detectable plasma concentrations were found only in 12
samples from 6 subjects.
[0168] Of the 12 detectable concentrations, only 2 were
significantly above the LLOQ, while others barely exceeded the LLOQ
of 5 ng/mL.
[0169] These two concentrations (11.1 and 48.0 ng/mL) were observed
in Subject 021 on Day 1, Hour 1 and just prior to the tenth dose on
Day 10, respectively.
[0170] It is to be noted that the 150 mg dose produced no
detectable concentrations.
[0171] Due to low MCC plasma levels across the dose range, there
were insufficient plasma data points above LLOQ for pharmacokinetic
analysis.
[0172] In the phase 2A study, after multiple dose oral
administrations, plasma concentrations of MCC were mostly below the
limit of quantification but with a dose dependent increase in the
number of samples, and number of subjects, with measurable plasma
concentrations.
[0173] Detectable plasma concentrations were found in 2/15 (13.3%)
subjects in the MCC 100 mg/day treatment group, 9/16 (56.3%)
subjects in the MCC 200 mg/day treatment group, and 13/17 (76.5%)
subjects in the MCC 400 mg/day treatment group.
[0174] Observable MCC concentrations ranged from 9.45 to 12.3 ng/mL
in the MCC 100 mg/day treatment group, 5.12 to 93.7 ng/mL in the
MCC 200 mg/day treatment group, and 5.32 to 84.9 ng/mL in the MCC
400 mg/day treatment group.
[0175] Of the detectable concentrations of MCC in all treatment
groups, the majority (35/41; 85.4%) were under 21 ng/mL.
[0176] Concentrations of MCC over 50 ng/mL were observed in only 2
subjects, one each in the 200 mg/day and 400 mg/day dosing
groups.
Urinary Excretion Data of MCC
[0177] Levels of MCC in the urine in the phase 1B-MD study were all
below the limit of quantification (LLOQ=5 ng/mL).
Fecal Concentration Data of MCC
[0178] Table 17 shows fecal concentrations from the 1B-MD study,
normalized to the 150 mg dose; fecal MCC averaged 916.0 .mu.g/g
(138.4-1768.9 .mu.g/g).
TABLE-US-00020 TABLE 17 Fecal concentrations of MCC in the phase
1B-MD study, normalized to a 150 mg dose. [MCC] Dose [MCC]
(normalized) Subject (mg) (.mu.g/g) (.mu.g/g) Range 150-450
415.1-5306.8 138.4-1768.9 Mean: 916.0 SD: 450.2
[0179] For the phase 2A study, in the MCC 100 mg/day treatment
group (n=11 samples sufficient), fecal MCC averaged 255.6 .mu.g/g
(range: 81.9-558.3 .mu.g/g) at the end of treatment. In the MCC 200
mg/day treatment group (n=9 samples sufficient), fecal MCC averaged
441.7 .mu.g/g (range: 11.7-786.7 .mu.g/g). In the MCC 400 mg/day
treatment group (n=13 samples sufficient), fecal MCC averaged
1433.3 .mu.g/g (range: 389.0-3974.8 .mu.g/g).
TABLE-US-00021 TABLE 18 Fecal concentrations of MCC at the end of
treatment in the phase 2A study. [MCC] [MCC] Dose range average
(mg/day) N (.mu.g/g) (.mu.g/g) 100 11 81.9-558.3 255.6 200 9
11.7-786.7 441.7 400 13 389.0-3974.8 1433.3
Conclusions
[0180] In summary, the present studies show that MCC is
well-tolerated after multiple oral doses up to 450 mg, achieves
high levels at the site of action, and shows promising results in
the treatment of C. difficile-associated diarrhea.
[0181] This study also found 1) there were no treatment-emergent
adverse events felt to be possibly drug related in either study, 2)
after multiple dose oral administrations, low MCC levels were
detected in plasma, most of which fell below the limit of
quantification. Consequential to low plasma concentrations, no
intact MCC was detected in the collected urine of the 1B-MD study.
3) by contrast, fecal levels in both studies were extremely high,
exceeding 10,000 times the MIC.sub.90 (0.125 .mu.g/mL) versus C.
difficile, 4) among 45 subjects treated with a full course of
therapy, only four subjects were considered failures prior to or at
the end of 10 days of therapy, 2 subjects in the 50-mg q12 hr and 2
subjects in the 100-mg q12 hr dose groups. No failures (0/16) were
noted in the 200-mg q12 hr dose, 5) recurrence was observed in only
2 subjects following successful treatment. Both recurred
approximately one month after the end of therapy, 6) although not
statistically significant, the median time-to-cessation of
diarrhea, showed a trend which suggested that higher doses may be
more efficacious. Time-to-cessation of diarrhea was determined to
be 5.5 days for the 50-mg q 12 hr dose group, 3.5 days for the
100-mg q 12 hr dose group, and 3.0 days for the 200-mg q 12 hr dose
group.
Example 3
MCC is Selectively Effective Against C. difficile In-Vivo, and does
not Affect Major Members of the Anaerobic Fecal Flora: Key to a
Lower Relapse Rate
[0182] To test the hypothesis that MCC is selectively active
in-vivo against C. difficile and could be relatively sparing of the
normal anaerobic fecal flora, quantitative stool cultures were
performed on serial stool samples obtained from patients entered
into a Phase 2A dose ranging clinical trial of MCC (now designated
MCC). Optimal antibiotic therapy of C. difficile diarrhea should
eradicate the vegetative forms of the pathogen, yet spare major
components of the normal flora presumed to be responsible for
colonization resistance.
Methods
[0183] Patients (n=32) were randomized to receive 50, 100 or 200 mg
twice daily of MCC for 10 days. No prior therapy was given to 24
patients; 8 receive 1 or 2 doses of standard therapy. As ecologic
controls, 7 additional patients were treated with vancomycin 125 mg
qid for 10 days. Fresh stool samples were cultured 10.sup.-2,4,6,8
for C. difficile vegetative and spore forms; fecal filtrates were
tested for cytotoxin B by cell assay. At study entry and day 10,
aerobic and anaerobic fecal flora cultures, diluted 10.sup.-3,5,7,9
were examined for major floral shifts. Since Bacteroides group
organisms are ubiquitously present and cultivable, this genera was
selected as a indicator of the integrity of the microbial
flora.
[0184] Detailed method shows as the following. [0185] 1) Single
center study in Calgary Health Region catchment area, population
.about.1 million [0186] 2) Randomized open label, dose ranging
Phase 2A study comparing 50 mg, 100 mg or 200 mg Q 12 hourly of MCC
for 10 days p. o. as therapy of CDAD. [0187] 3) Following
completion of the trial recruitment, a separate ecology control
group of patients who otherwise would be eligible for the trial
were treated with Vancomycin 125 mg QID for 10 days as a
treatment/ecologic control. [0188] 4) Mild to moderate CDAD: >3
but <12 diarrheal samples/24 hours at study entry, positive C.
difficile toxin assay, fever <39 degrees C., WBC <30,000/mm3,
no vomiting, no severe abdominal discomfort [0189] 5) Primary CDAD
or first relapse episode only. [0190] 6) Treatment naive if
possible. The protocol allowed up to 3 prior doses of standard
therapy, but for this evaluation, a maximum of 2 doses of standard
therapy was allowed. In this study population, 24 patients were
treatment naive [0191] 7) No concomitant parenteral antibiotic
therapy for any condition. [0192] 8) Serial stool samples: in
addition to the original diagnostic sample, a repeat collection of
stool >5 grams (10-30 grams usually) was obtained at study
entry, at day 4, 7, 10, 14, 21, 28 and 42 days after study entry
[0193] 9) For this report, results of day 0 and day 10 stools are
compared for changes in C. difficile counts and in counts of major
genera of the normal colonic flora. [0194] 10) C. difficile
quantitative counts and fecal filtrate concentrations of C.
difficile cytotoxin B by HeLa cell assay were determined with
freshly passed samples as refrigeration is deleterious to
determination of quantitative counts of C. difficile. [0195] 11)
Since Bacteroides group organisms are considered to be uniformly
present in subjects and in high counts, and is likely one of the
major components of the normal flora conferring `colonization
resistance`, this group was used as an index of suppression of the
anaerobic fecal flora. For patients who failed to show return of
the Bacteroides group species at 10 days, subsequent samples were
processed to document time of return of this group. If samples were
not immediately processed, aliquots were frozen at -80 degrees C.
with 15% glycerol/Brain Heart Infusion Broth for subsequent
processing. [0196] 12) Media and methods for anaerobic flora
cultures are based on the Wadsworth-KTL Anaerobic Manual, 6th ed,
2002. C. difficile counts were determined by dilution of the sample
10.sup.-2,4,6,8/gram stool wet weight on CCFA agar. Spore counts
were determined by treating an aliquot of stool with an equal
volume of 100% ethyl alcohol.times.1 hour, centrifuged, washed
twice and resuspended for quantitative counts. [0197] 13) Normal
flora cultures were quantified by dilution 10.sup.-3,5,7,9 using
MacConkey, BAP, m-Enterococcus agar, Lab M anaerobic blood agar,
BAP, BBE, KVLB, PEA agars incubated for 48 hours before initial
inspection, and further incubated for up to 7 days. [0198] 14) For
vancomycin ecologic controls, vancomycin fecal filtrate
concentrations were determined in triplicate by bioassay using a C.
perfringens as the indicator organism. [0199] 15) Differences in
microbial counts were determined after log.sub.10 transformation
using wilcoxon matched.
Results
[0200] At study entry, mean log.sub.10 CFU.+-.SD vegetative counts
of C. difficile (all MCC patients) were 6.8.+-.3.6, range 2-10.95;
at day 10, with the exception of one patient receiving 50 mg, all
other patients had C. difficile quantitative counts reduced <2
log.sub.10/gm feces. Vancomycin was similarly effective. At study
entry, Bacteroides group counts were <3, 3-8, & 8.5-10
log.sub.10 CFU/gm in 1/3 each of patients, with normal counts being
>11. Shifts in the Bacteroides group are shown table 19.
TABLE-US-00022 TABLE 19 Mean .+-. SD of log.sub.10 CFU of
Bacteroides group counts/gm feces wet weight MCC, 50 mg MCC, 100 mg
MCC, 200 mg Vancomycin (n = 10) (n = 8) (n = 11) (n = 7) Day 0 6.64
.+-. 2.82 6.64 .+-. 2.82 7.04 .+-. 2.87 7.39 .+-. 2.67 Day 10 8.23
.+-. 2.60 6.30 .+-. 2.53 7.34 .+-. 3.06 3.62** .+-. 1.90 p* 0.11
0.44 0.56 0.03 *wilcoxon matched pairs signed-ranks test, 2 tailed;
**counts <3 log.sub.10 = 2.90
[0201] The following figures further illustrate the results from
the study.
Conclusions
[0202] Based on quantitative Bacteroides group counts, patients
with C. difficile diarrhea have variably impaired normal flora at
study entry, with approximately 1/3 in the 3 log 10 CFU/gm range,
1/3 in counts of 4-7 log 10 CFU, and the remainder with higher
counts (none in the normal range of 11-12 log 10 CFU). All dosages
of MCC appeared to reduce counts of C. difficile, as did
vancomycin. A dose dependent reduction in Bacteroides counts with
increasing dosages of MCC was not observed. Vancomycin severely
impairs Bacteroides counts during therapy and although most
patients recover their counts, a minority have prolonged
absence.
[0203] Based on these data and clinical outcomes showing a high
response rate accompanied by a low relapse rate, it would appear
that the 200 mg dose of MCC would be an appropriate dosage to
undergo further clinical investigation.
[0204] The invention is not limited by the embodiments described
above which are presented as examples only but can be modified in
various ways within the scope of protection defined by the appended
patent claims
* * * * *